We have generated conditional glucocorticoid receptor (GR) knockout mice, which have been crossed to lck-Cre animals to produce mice that lack GR expression in thymocytes and T cells (GRlck-Cre). These mice have a modest reduction (about 40%) in the number of double positive (DP) and single positive (SP) thymocytes. Introduction of a transgenic T cell receptor (TCR) that has differing affinities for different MHC-encoded class II molecules revealed that the higher the avidity for self, the greater the reduction in thymocyte number in the GR KO mice, indicating that the thymocytes are undergoing increased negative selection in the absence of GR signaling. If the TCR repertoire, the range of receptors expressed after selection, is indeed affected by glucocorticoids, there should be changes in the specificity of immune responses. We have found that mature T cells from the conditional GR-null mice proliferate normally to mitogens or to TCR cross-linking, but poorly to alloantigen. Moreover, immunization with peptide antigens results in a poor T cell recall response. Strikingly, if the TCR repertoire is fixed by introducing alpha/beta TCR transgenes, the proliferative response of GRlci-Cre T cells to antigen in normal, in vitro and in vivo. Deep sequencing of TCRbeta CD3 regions, which have the largest contribution to TCR specificity, found that there was a significant difference between, but not within, groups of wild type and GRlci-Cre naive T cells. These results demonstrate that exposure to glucocorticoids in the thymus is a critical event in shaping the T cell repertoire, and thus the ability to respond to foreign pathogens. We have now made mice in which the enzyme responsible for corticosterone production, CYP11B1, encoding steroid 11beta-hydroxylase, is floxed. We have crossed these mice onto Cre-expressing animals to knockout glucocorticoid production in thymic epithelial cells, allowing us to test the hypothesis that local glucocorticoid production is important for thymocyte development. We have found that the T cell response to alloantigen is blunted, just as it is in the GRlck-Cre mice. Furthermore, the response to infection with the virus LCMV show changes in the fine-specificity of the TCRs uses (as assessed by tetramer staining). We have also found that the constitutive expression of glucocorticoid-responsive genes is reduced to baseline in these mice, suggesting that the bulk of the bioavailable glucocorticoids are produced locally. In another series of studies we found that if one uses a cell fixative plus a detergent (to permeabilize the membrane), glucocorticoid receptors that are ligand bound and chromatin-associated will be cross linked to adjacent chromatin. As a result, the GR (and other nuclear receptors) can be used as biosensors that can quantitate ambient ligand concentration. Use this perm-fix technique we have been able to identify for the first time the effects of paracrine hormone production in the thymus. We have found that DP thymocytes that have been triggered by self antigen are in a high glucocorticoid environment (approximately threefold higher than other thymocytes) due to corticosterone production by thymic epithelial cells. This assay allows the detection and quantification of hormone exposure in tissues at the single cell level.